This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The formation of carbohydrate-protein complexes is important in a variety of processes involving the interaction of a cell with its environment. These processes include natural ones such as cell differentiation, cell aggregation, and cell signaling;they also include disease processes such as viral infection, malignancy, and unwanted inflammation. The development of drugs to moderate natural processes and inhibit disease processes begins with accurate structural models for oligosaccharide-protein interactions. These models have been difficult to get by traditional Nuclear Magnetic Resonance (NMR) approaches because of the dearth of short-distance NOE contacts in oligosaccharide-protein complexes. We are developing a number of experiments and analysis tools that allow retrieval of structural information when traditional NMR approaches fail. This particular project combines new isotopic labeling strategies developed in the Moremen group with two new types of NMR-observable structural data. One type comes from residual dipolar couplings of labeled 15N-1H pairs in various amide bonds. These give orientational constraints on bond vectors. The other type comes from long range paramagnetic spin relaxation perturbation of labeled sites by synthesized nitroxide-carrying analogs of protein ligands. These give long-range distance constraints between the bound ligand and the labeled sites. The sialyltransferase, ST6Gal-I, has been the primary target motivating this work. This transferase is essential to the synthesis of sialic acid terminated glycans on many glycoproteins, including those involved in the activation of B-cells. It was first cloned more than 18 years ago, but it continues to resist attempts at structural characterization by traditional means. A second target, the fucosyltransferase, FucT-III, is also structurally uncharacterized and equally important biologically. Both are representative of the many mammalian proteins that are best expressed in non-bacterial hosts.